Xenobiotic-Metabolizing Enzymes

代谢组学是分析存在于液体、组织或细胞中的代谢物的一种理想方法，并且正在发展成为药物研究和开发的有用工具。各种分析平台[UPLC-ESI-QTOFMS，气相色谱- ms和核磁共振]和多变量数据分析[如主成分分析（PCA），偏最小二乘判别分析（PLS-DA）和正交投影到潜在结构判别分析（OPLS-DA）]已被应用于基于代谢组学的异种生物代谢研究。利用基于UPLC-ESI-QTOFMS的代谢组学，测定了对乙酰氨基酚、氨基黄素、茴香碱、褪黑素、2-氨基-1-甲基-6-苯咪唑[4,5-b]吡啶和异环磷酰胺/环磷酰胺、硫代tepa、普鲁卡因酰胺和诺斯卡平的代谢图谱。代谢组学已被证明是研究药物和致癌物代谢以及寻找药物毒性和疗效生物标志物的有力方法。当与P450基因敲除和人源化小鼠结合使用时，这种方法尤其有价值。这项技术也被用于寻找可用于癌症诊断和癌症治疗监测的疾病生物标志物。我们的大多数筛选研究都是使用小鼠癌症模型来识别生物标志物。一旦确定，生物标志物将使用来自人类患者的样本进行验证。采用代谢组学方法对诺斯卡平的代谢进行了研究。生成了代谢图，并研究了诺斯卡平的生物活性。采用UPLC-ESI-QTOFMS对诺斯卡平处理小鼠体外培养液、尿液和粪便样品进行分析。利用重组药物代谢酶鉴定参与诺斯卡平代谢的酶。肝脏还原型谷胱甘肽（GSH）水平和血清生化测定诺斯卡平的反应性代谢物。在小鼠灌胃后检测到诺斯高平的几种新的I期代谢物，包括一个n -去甲基化代谢物，两个羟基化代谢物，一个去甲基化和亚二氧基裂解的代谢物，以及一个双去甲基化代谢物。此外，还检测到几种新的葡糖苷类化合物，并通过MS/MS片段学分析了它们的结构。重组酶筛选表明，几种细胞色素P450、含黄素单加氧酶1和udp -葡萄糖醛基转移酶UGT1A1、UGT1A3、UGT1A9和UGT2B7参与诺斯卡平代谢。在体外谷胱甘肽捕获揭示了通过进一步氧化儿茶酚代谢物形成的对醌反应中间体的存在。然而，诺斯卡平的这种生物活化过程不会在体内发生。与此结果类似，肝脏中谷胱甘肽水平的改变和血清生化没有显示肝损伤的证据，这表明，至少在小鼠中，诺斯卡平不会通过生物激活诱导肝毒性。全面的代谢图谱和生物活性评价为诺斯卡平作为抗肿瘤药物的开发提供了重要信息。代谢组学还用于确定普鲁卡因胺的代谢，普鲁卡因胺是一种I型抗心律失常药物，用于治疗各种心房和室性心律失常。据报道，长期使用普鲁卡因胺治疗可能导致25-30%的患者出现红斑狼疮。有趣的是，普鲁卡因胺在小鼠模型中不会诱发红斑狼疮。为了探究普鲁卡因胺在人和小鼠模型中这种副作用的差异，我们使用UPLC-ESI-QTOFMS对普鲁卡因胺处理的人、cyp2d6人源化小鼠和野生型小鼠的尿液样本进行了代谢组学分析。13种尿普鲁卡因胺代谢物，包括9种新的代谢物，来源于p450依赖、fmo依赖的氧化和酰化反应，并进行了结构鉴定。人CYP2D6人源化小鼠体内普鲁卡因胺的代谢以及与微粒体和重组p450的体外培养表明，人CYP2D6在普鲁卡因胺的代谢中起主要作用。人类和小鼠之间药物n -酰化和n -氧化的显著差异在很大程度上解释了普鲁卡因胺代谢的种间差异。人体中由FMO1和FMO3产生的新型n -氧化物代谢物的显著水平可能与普鲁卡因酰胺诱导的系统性红斑狼疮的发展有关。基于代谢组学研究的观察结果为理解普鲁卡因酰胺诱导的人类狼疮以及p450和FMOs对普鲁卡因酰胺n氧化的影响提供了线索。细胞色素P450也在人类胎儿肝脏中表达，并可能在胎儿毒性中起作用。CYP3A7是人胎肝中主要表达的细胞色素P450，占胎肝总CYP的30-50%，占胎肝总CYP3A含量的87-100%。然而，缺乏啮齿动物模型限制了CYP3A7调控和功能的研究。因此，我们利用表达人PXR和CYP3A4/7的双转基因小鼠（Tg3A4/7-hPXR）来研究CYP3A7的调控和功能。在14.5 d龄胚胎至8.5 d龄新生儿小鼠中监测CYP3A7的表达；胚胎中CYP3A7 mRNA的表达在出生前升高，新生儿中CYP3A7 mRNA的表达在出生前降低。这与观察到的CYP3A7蛋白水平和CYP3A7介导的脱氢表雄酮16 α -羟化酶活性的发育调节一致。这种发育通量也与先前研究CYP3A7在发育中的人肝脏中的表达一致。利用Tg3A4/7-hPXR小鼠的肝母细胞进一步研究CYP3A7的调控。地塞米松、皮质醇、皮质酮、可的松等糖皮质激素均可诱导CYP3A7 mRNA的表达，而成人肝脏中PXR的激活剂、CYP3A4的诱诱剂利福平对CYP3A7的表达无影响。基于细胞的启动子荧光素酶和染色质免疫沉淀实验进一步证实了糖皮质激素受体介导的CYP3A7启动子的控制。这些发现表明，CYP3A7主要通过糖皮质激素受体在小鼠肝脏中受到糖皮质激素的发育调节。因此，Tg3A4/7-hPXR小鼠模型可以作为研究CYP3A7调控和功能的潜在工具。

Metabolomics, is an ideal approach for analysis of the metabolites present in fluids, tissues or cells, and is evolving as a useful tool in drug research and development. Various analytical platforms [UPLC-ESI-QTOFMS, gas GC-MS and nuclear magnetic resonance] and multivariate data analysis [such as principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA)] have been applied in metabolomic-based xenobiotic metabolism studies. Using UPLC-ESI-QTOFMS based metabolomics, the metabolic maps have been determined for acetaminophen, aminoflavin, arecoline, melatonin, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and ifosfamide/cyclophosphamide, thioTEPA, procainamide, and noscarpine. Metabolomics has proven to be a powerful method for study of drug and carcinogen metabolism and to search for biomarkers of drug toxicity and efficacy. This methodology is especially valuable when combined with the use of P450 knockout and humanized mice. This technique is also being used to search for disease biomarkers that can be employed for diagnosis of cancer and monitoring of cancer therapy. Most of our screening studies are carried out using mouse cancer models to identify the biomarkers. Once identified, the biomarkers will be validated using samples from human patients.The metabolism of noscapine, a promising anti-tumor agent, was examined by metabolomics. A metabolic map was generated and the bioactivation of noscapine investigated. UPLC-ESI-QTOFMS was used to analyze in vitro incubation mixtures, urine and feces samples from mice treated with noscapine. Recombinant drug-metabolizing enzymes were employed to identify those involved in noscapine metabolism. Hepatic reduced glutathione (GSH) levels and serum biochemistry were also carried out to determine reactive metabolites of noscapine. Several novel phase I metabolites of noscapine were detected after oral gavage of mice, including an N-demethylated metabolite, two hydroxylated metabolites, one metabolite undergoing both demethylation and cleavage of the methylenedioxy group, and a bis-demethylated metabolite. Additionally, several novel glucuronides were detected, and their structures elucidated through MS/MS fragmentology. Recombinant enzymes screening showed the involvement of several cytochromes P450, flavin-containing monooxygenase 1 and the UDP-glucuronosyltransferases UGT1A1, UGT1A3, UGT1A9 and UGT2B7, in noscapine metabolism. In vitro glutathione trapping revealed the existence of an ortho-quinone reactive intermediate formed through further oxidation of a catechol metabolite. However, this bioactivation process of noscapine does not occur in vivo. Similar to this result, altered glutathione levels in liver and serum biochemistry revealed no evidence for hepatic damage thus indicating that, at least in mice, noscapine did not induce hepatotoxicity through bioactivation. A comprehensive metabolic map and bioactivation evaluation provides important information for the development of noscapine as an anti-tumor drug.Metabolomics was also used to determine the metabolism of procainamide, a type I antiarrhythmic agent, used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25-30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using UPLC-ESI-QTOFMS was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice aswell as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.Cytochromes P450 are also expressed in fetal livers of humans and may have a role of fetal toxicity. CYP3A7 is the predominant cytochrome P450 expressed in human fetal liver, accounting for 30-50% of the total CYP in fetal liver and 87-100% of total fetal hepatic CYP3A content. However, the lack of a rodent model limits the investigation of CYP3A7 regulation and function. Hence, double-transgenic mice expressing human PXR and CYP3A4/7 (Tg3A4/7-hPXR) were used to investigate the regulation and function of CYP3A7. Expression of CYP3A7 was monitored in mice that ranged in age from 14.5-d-old embryos to 8.5-d-old newborns; expression of CYP3A7 mRNA was increased before birth in the embryos and decreased after birth in the newborns. This is consistent with the observed developmental regulation of CYP3A7 protein levels and CYP3A7-mediated dehydroepiandrosterone 16alpha-hydroxylase activities. This developmental flux is also in agreement with previous studies that have investigated the expression of CYP3A7 in developing human liver. The regulation of CYP3A7 was further studied using hepatoblasts from the Tg3A4/7-hPXR mice. Glucocorticoids, including dexamethasone, cortisol, corticosterone, and cortisone all induced the expression of CYP3A7 mRNA, whereas rifampicin, an activator of PXR and an inducer of CYP3A4 in adult liver, had no effect on CYP3A7 expression. Cell-based promoter luciferase and chromatin immunoprecipitation assays further confirmed glucocorticoid receptor-mediated control of the CYP3A7 promoter. These findings indicate that CYP3A7 is developmentally regulated in mouse liver primarily by glucocorticoids through the glucocorticoid receptor. The Tg3A4/7-hPXR mouse model could therefore potentially serve as a tool for investigating CYP3A7 regulation and function.